Vpu enhances retrovirus particle release from the plasma membrane at a stage following classical particle budding. Vpu inhibits a potent cellular restriction to release that was identified in 2008 as BST2 or tetherin. The overall goals of this project are to define the mechanism of restriction of particle release by tetherin, and the mechanism by which Vpu overcomes tetherin-mediated restriction. Recycling pathways, the actin-associated cytoskeleton, and the role of tetherin in HIV-infected macrophages are specific areas of focus in this project. Experiments in Aim 1 will define the role of cellular recycling pathways on tetherin-mediated restriction of particle release. Dominant-negative inhibitors of specific steps in endocytic trafficking and recycling will be employed to dissect the trafficking of tetherin and correlate this with restriction. Experiments in Aim 2 examine the role of tetherin in HIV-infected human macrophages. The potential role of tetherin in forming the HIV particle-enriched intracellular compartment in macrophages will be addressed. In Aim 3, the role of RICH2, Ezrin, and the actin-associated cytoskeleton in tetherin-mediated restriction will be examined. The role of the coiled-coil domain on the extracellular portion of tetherin will be analyzed using a null mutant. Together, these studies will provide important new information related to the cellular biology of tetherin and the paths through which Vpu acts to overcome tetherin-mediated retention of viral particles.